Immunotherapy of autoimmune disorders using antibodies which target b-cells

ABSTRACT

Antibodies that bind with a B-cell antigen provide an effective means to treat autoinmune disorders. Antibodies and fragments, which may be conjugated or naked, are used alone or in multimodal therapies. The antibodies may be bispecific antibodies which may be produced recombinantly as fusion proteins, or as hybrid, polyspecific antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/104,594, filed Apr. 13, 2005, which is a continuation of U.S. patentapplication Ser. No. 09/590,284, filed Jun. 9, 2000, now U.S. Pat. No.7,074,403, which claims priority to U.S. Provisional Application No.60/138,284, filed Jun. 9, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to immunotherapeutic methods for treatingautoimmune disorders. In particular, this invention is directed tomethods for treating autoimmune disorders by administering antibodiesthat bind to a B-cell antigen, such as the CD22, CD20, CD19, and CD74 orHLA-DR antigen. The antibodies are administered alone or in combination,and may be naked or conjugated to a drug, toxin or therapeuticradioisotope. Bispecific antibody fusion proteins which bind to theB-cell antigens can be used according to the present invention,including hybrid antibodies which bind to more than one B-cell antigen.The present invention also is directed to multimodal therapeutic methodsin which the antibody administration is supplemented by administrationof other therapeutic modalities.

2. Background

Antibodies against the CD20 antigen have been investigated for thetherapy of B-cell lymphomas. For example, a chimeric anti-CD20 antibody,designated as “IDEC-C2B8,” has activity against B-cell lymphomas whenprovided as unconjugated antibodies at repeated injections of dosesexceeding 500 mg per injection. Maloney et al., Blood 84:2457 (1994);Longo, Curr. Opin. Oncol. 8:353 (1996). About 50 percent ofnon-Hodgkin's patients, having the low-grade indolent form, treated withthis regimen showed responses. Therapeutic responses have also beenobtained using ¹³¹I-labeled B1 anti-CD-20 murine monoclonal antibodywhen provided as repeated doses exceeding 600 mg per injection. Kaminskiet al., N. Engl. J. Med. 329:459 (1993); Press et al., N. Engl. J. Med.329:1219 (1993); Press et al., Lancet 346:336 (1995). However, theseantibodies, whether provided as unconjugated forms or radiolabeledforms, have shown only modest activity in patients with the moreprevalent and lethal form of B-cell lymphoma, the intermediate oraggressive type.

Autoimmune diseases are a class of diseases associated with a B-celldisorder. Examples include immune-mediated thrombocytopenias, such asacute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, myasthenia gravis, lupus nephritis, lupuserythematosus, and rheumatoid arthritis. The most common treatments arecorticosteroids and cytotoxic drugs, which can be very toxic. Thesedrugs also suppress the entire immune system, can result in seriousinfection, and have adverse affects on the liver and kidneys. Othertherapeutics that have been used to treat Class III autoimmune diseasesto date have been directed against T-cells and macrophages. A needremains for more effective methods of treating autoimmune diseases,particularly Class III autoimmune diseases.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide amethod for treating autoimmune diseases using antibody to a B-cellantigen.

It is another object of the invention is to use comparatively low dosesof a naked antibody to a B-cell antigen, preferably to CD22 antigen, ora combination of naked antibodies to a CD22 antigen and another B-cellantigen, preferably CD20 and/or CD74.

Yet another object of the invention is to use a combination of one ormore naked antibodies to B-cell antigens and/or antibodies to B-cellantigens which are conjugated to drugs, toxins or therapeuticradioisotopes.

It is a further object of this invention to provide multimodal methodsfor treatment of autoimmune diseases in which a naked or conjugatedantibody to a B-cell antigen is supplemented with the administration ofother therapeutic modalities, such as those directed against T-cells,plasma cells and macrophages.

These and other objects are achieved, in accordance with one embodimentof the present invention, by the provision of a method of treating anautoimmune disease, comprising the step of administering to a subjecthaving an autoimmune disease an antibody to a B-cell antigen and apharmaceutically acceptable carrier.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION

1. Overview

B-cell clones that bear autoantibody Ig-receptors are present in normalindividuals. Autoimmunity results when these B-cells become overactive,and mature to plasma cells that secrete autoantibody. In accordance withthe present invention, autoimmune disorders can be treated byadministering an antibody that binds to a B-cell antigen, such as theCD22, CD20, CD19, and CD74 or HLA-DR antigen. In one embodiment,comparatively low doses of an entire, naked antibody or combination ofentire, naked antibodies are used. In other embodiments, conjugates ofsuch antibodies with drugs, toxins or therapeutic radioisotopes areuseful. Bispecific antibody fusion proteins which bind to the B-cellantigens can be used according to the present invention, includinghybrid antibodies which bind to more than one B-cell antigen. Preferablythe bispecific and hybrid antibodies additionally target a T-cell,plasma cell or macrophage antigen. The present invention also isdirected to multimodal therapeutic methods in which the antibodyadministration is supplemented by administration of other therapeuticmodalities.

2. Definitions

In the description that follows, and in documents incorporated byreference, a number of terms are used extensively. The followingdefinitions are provided to facilitate understanding of the invention.

A structural gene is a DNA sequence that is transcribed into messengerRNA (mRNA) which is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

A promoter is a DNA sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent when the promoter isa constitutive promoter.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe genomic DNA of an organism. For example, a cloned antibody gene is aDNA fragment that has been separated from the genomic DNA of a mammaliancell. Another example of an isolated DNA molecule is a chemicallysynthesized DNA molecule that is not integrated in the genomic DNA of anorganism.

An enhancer is a DNA regulatory element that can increase the efficiencyof transcription, regardless of the distance or orientation of theenhancer relative to the start site of transcription.

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from a mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

The term expression refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

As used herein, antibody encompasses naked antibodies and conjugatedantibodies and antibody fragments, which may be monospecific ormultispecific. It includes both polyclonal and monoclonal antibodies, aswell as certain recombinant antibodies, such as chimeric and humanizedantibodies and fusion proteins.

A chimeric antibody is a recombinant protein that contains the variabledomains and complementary determining regions derived from a rodentantibody, while the remainder of the antibody molecule is derived from ahuman antibody.

Humanized antibodies are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

Human antibodies are antibodies that either are isolated from humans andthen grown out in culture or are made using animals whose immune systemshave been altered so that they respond to antigen stimulation byproducing human antibodies.

As used herein, a therapeutic agent is a molecule or atom, which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,enzymes, hormones, cytokines, immunomodulators, boron compounds andtherapeutic radioisotopes. Preferred therapeutic radioisotopes includebeta, alpha, and Auger emitters, with a kev range of 80-500 kev.Exemplary therapeutic radioisotopes include ¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y,¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu, and ²¹¹At.

A naked antibody is an entire antibody which is not conjugated with atherapeutic agent. Naked antibodies include both polyclonal andmonoclonal antibodies, as well as certain recombinant antibodies, suchas chimeric and humanized antibodies.

A conjugated antibody is an antibody or antibody fragment that isconjugated to a therapeutic agent.

A multispecific antibody is an antibody which can bind simultaneously toat least two targets which are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. One specificity would be for aB-cell antigen or epitope.

A bispecific antibody is an antibody which can bind simultaneously totwo targets which are of different structure. Bispecific antibodies(bsAb) and bispecific antibody fragments (bsFab) have at least one armthat specifically binds to a B-cell antigen or epitope and at least oneother arm that specifically binds a targetable conjugate.

A fusion protein is a recombinantly produced antigen-binding molecule inwhich two or more different single-chain antibody or antibody fragmentsegments with the same or different specificities are linked. A varietyof bispecific fusion proteins can be produced using molecularengineering. In one form, the bispecific fusion protein is monovalent,consisting of, for example, a scFv with a single binding site for oneantigen and a Fab fragment with a single binding site for a secondantigen. In another form, the bispecific fusion protein is divalent,consisting of, for example, an IgG with two binding sites for oneantigen and two scFv with two binding sites for a second antigen.

3. Production of Monoclonal Antibodies, Humanized Antibodies, PrimateAntibodies and Human Antibodies

Anti-CD20, anti-CD22, anti-CD19, and anti-CD74 antibodies are knowngenerally to those of skill in the art. See, for example, Ghetie et al.,Cancer Res. 48:2610 (1988); Hekman et al., Cancer Immunol. Immunother.32:364 (1991); Kaminski et al., N. Engl. J. Med. 329:459 (1993); Presset al., N. Engl. J. Med. 329:1219 (1993); Maloney et al., Blood 84:2457(1994); Press et al., Lancet 346:336 (1995); Longo, Curr. Opin. Oncol.8:353 (1996). More particularly, rodent monoclonal antibodies to CD22,CD20, CD19, or CD74 antigens can be obtained by methods known to thoseskilled in the art. See generally, for example, Kohler and Milstein,Nature 256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS INIMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”]. Briefly, monoclonal antibodies can be obtained by injectingmice with a composition comprising the antigen, verifying the presenceof antibody production by removing a serum sample, removing the spleento obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen that was injected, culturing theclones that produce antibodies to the antigen, and isolating theantibodies from the hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Suitable amounts of well-characterized antigen for production ofantibodies can be obtained using standard techniques. As an example,CD22 can be immunoprecipitated from B-lymphocyte protein using thedeposited antibodies described by Tedder et al., U.S. Pat. No. 5,484,892(1996).

Alternatively, CD22, CD20, CD19, or CD74 antigen proteins can beobtained from transfected cultured cells that overproduce the antigen ofinterest. Expression vectors that comprise DNA molecules encoding eachof these proteins can be constructed using published nucleotidesequences. See, for example, Wilson et al., J. Exp. Med. 173:137 (1991);Wilson et al., J. Immunol. 150:5013 (1993). As an illustration, DNAmolecules encoding CD22 can be obtained by synthesizing DNA moleculesusing mutually priming long oligonucleotides. See, for example, Ausubelet al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to8.2.13 (1990) [“Ausubel”]. Also, see Wosnick et al., Gene 60:115 (1987);and Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rdEdition, pages 8-8 to 8-9 (John Wiley & Sons, Inc. 1995). Establishedtechniques using the polymerase chain reaction provide the ability tosynthesize genes as large as 1.8 kilobases in length. Adang et al.,Plant Molec. Biol. 21:1131 (1993); Bambot et al., PCR Methods andApplications 2:266 (1993); Dillon et al., “Use of the Polymerase ChainReaction for the Rapid Construction of Synthetic Genes,” in METHODS INMOLECULAR BIOLOGY, Vol. 15: PCR PROTOCOLS: CURRENT METHODS ANDAPPLICATIONS, White (ed.), pages 263-268, (Humana Press, Inc. 1993).

In a variation of this approach, monoclonal antibody can be obtained byfusing myeloma cells with spleen cells from mice immunize with a murinepre-B cell line stably transfected with cDNA which encodes the antigenof interest. See Tedder et al., U.S. Pat. No. 5,484,892 (1996).

One example of a suitable murine anti-CD22 monoclonal antibody is theLL2 (formerly EPB-2) monoclonal antibody, which was produced againsthuman Raji cells derived from a Burkitt lymphoma. Pawlak-Byczkowska etal., Cancer Res. 49:4568 (1989). This monoclonal antibody has anIgG_(2α) isotype, and the antibody is rapidly internalized into lymphomacells. Shih et at, Int. J. Cancer 56:538 (1994). Immunostaining and invivo radioimmunodetection studies have demonstrated the excellentsensitivity of LL2 in detecting B-cell lymphomas. Pawlak-Byczkowska etal., Cancer Res. 49:4568 (1989); Murthy et al., Eur. J. Nucl. Med.19:394 (1992). Moreover, ^(99m)Tc-labeled LL2-Fab′ fragments have beenshown to be useful in following upstaging of B-cell lymphomas, while¹³¹I-labeled intact LL2 and labeled LL2 F(ab′)₂ fragments have been usedto target lymphoma sites and to induce therapeutic responses. Murthy etal., Eur. J. Nucl. Med. 19:394, (1992); Mills et al., Proc. Am. Assoc.Cancer Res. 34:479 (1993) [Abstract 2857]; Baum et al., Cancer 73(Suppl. 3):896 (1994); Goldenberg et al., J. Clin. Oncol. 9:548 (1991).Furthermore, Fab′ LL2 fragments conjugated with a derivative ofPseudomonas exotoxin has been shown to induce complete remissions formeasurable human lymphoma xenografts growing in nude mice. Kreitman etal., Cancer Res. 53:819 (1993). An example of an anti-CD74 antibody isthe LL1 antibody.

In an additional embodiment, an antibody of the present invention is achimeric antibody in which the variable regions of a human antibody havebeen replaced by the variable regions of a rodent anti-CD22 antibody.The advantages of chimeric antibodies include decreased immunogenicityand increased in vivo stability.

Techniques for constructing chimeric antibodies are well known to thoseof skill in the art. As an example, Leung et al., Hybridoma 13:469(1994), describe how they produced an LL2 chimera by combining DNAsequences encoding the V_(κ) and V_(H) domains of LL2 monoclonalantibody with respective human κ and IgG, constant region domains. Thispublication also provides the nucleotide sequences of the LL2 light andheavy chain variable regions, V_(η) and V_(H), respectively.

In another embodiment, an antibody of the present invention is asubhuman primate antibody. General techniques for raisingtherapeutically useful antibodies in baboons may be found, for example,in Goldenberg et al., international patent publication No. WO 91/11465(1991), and in Losman et al., Int. J. Cancer 46: 310 (1990).

In yet another embodiment, an antibody of the present invention is a“humanized” monoclonal antibody. That is, mouse complementaritydetermining regions are transferred from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, followed bythe replacement of some human residues in the framework regions of theirmurine counterparts. Humanized monoclonal antibodies in accordance withthis invention are suitable for use in therapeutic methods. Generaltechniques for cloning murine immunoglobulin variable domains aredescribed, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988),Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437(1992), and Singer et al., J. Immun. 150:2844 (1993). The publication ofLeung et al., Mol. Immunol. 32:1413 (1995), describes the constructionof humanized LL2 antibody.

In another embodiment, an antibody of the present invention is a humanmonoclonal antibody. Such antibodies are obtained from transgenic micethat have been “engineered”. to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994).

4. Production of Bispecific Antibodies

The present invention also may employ a bispecific antibody (bsAb) orantibody fragment (bsFab) having at least one arm that specificallybinds to a B-cell antigen and at least one other arm that specificallybinds a targetable conjugate. The targetable conjugate comprises acarrier portion which comprises or bears at least one epitope recognizedby at least one arm of the bispecific antibody or antibody fragment. Ina preferred embodiment, the epitope is a hapten. In an alternativeembodiment, the epitope is a part of the carrier. Examples ofrecognizable haptens include, but are not limited to, chelators, such asDTPA, fluorescein isothiocyanate, vitamin B-12 and other moieties towhich specific antibodies can be raised. The carrier portion also may beconjugated to a variety of agents. Examples of conjugated agentsinclude, but are not limited to, metal chelate complexes, drugs, toxinsand other effector molecules, such as cytokines, lymphokines,chemokines, immunomodulators, radiosensitizers, asparaginase, carboranesand radioactive halogens. Additionally, enzymes useful for activating aprodrug or increasing the target-specific toxicity of a drug can beconjugated to the carrier. Thus, the use of bispecific antibodies andfragments which have at least one arm that specifically binds atargetable conjugate allows a variety of therapeutic and diagnosticapplications to be performed without raising new bsAb for eachapplication.

The present invention encompasses antibodies and antibody fragments. Theantibody fragments are antigen binding portions of an antibody, such asF(ab′)2, F(ab)2, Fab′, Fab, and the like. The antibody fragments bind tothe same antigen that is recognized by the intact antibody. For example,an anti-CD22 monoclonal antibody fragment binds to an epitope of CD22.The bsAb of the present invention include, but are not limited to,IgG×IgG, IgG×F(ab′)2, IgG×Fab′, IgG×scFv, F(ab′)2×F(ab′)2, Fab′×F(ab′)2,Fab′×Fab′, Fab′×scFv and scFv×scFv bsmabs. Also, species such asscFv×IgG×scFv and Fab′×IgG×Fab′, scFv×F(ab′)2×scFv and Fab′×F(ab′)2×Fab′are included.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments, “Fv” fragments, consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy chain variable regions are connectedby a peptide linker (“sFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion.

5. Production of Fusion Proteins

Another method for producing bsAbs is to engineer recombinant fusionproteins linking two or more different single-chain antibody or antibodyfragment segments with the needed dual specificities. See, e.g., Colomaet al., Nature Biotech. 15:159-163, 1997. A variety of bispecific fusionproteins can be produced using molecular engineering. In one form, thebispecific fusion protein is monovalent, consisting of, for example, ascFv with a single binding site for one antigen and a Fab fragment witha single binding site for a second antigen. In another form, thebispecific fusion protein is divalent, consisting of, for example, anIgG with two binding sites for one antigen and two scFv with two bindingsites for a second antigen.

Functional bispecific single-chain antibodies (bscAb), also calleddiabodies, can be produced in mammalian cells using recombinant methods.See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995. Forexample, bscAb are produced by joining two single-chain Fv fragments viaa glycine-serine linker using recombinant methods. The V light-chain(VL) and V heavy-chain (VH) domains of two antibodies of interest areisolated using standard PCR methods. The VL and VH cDNA's obtained fromeach hybridoma are then joined to form a single-chain fragment in atwo-step fusion PCR. The first PCR step introduces the (Gly4-Ser1)3linker, and the second step joins the VL and VH amplicons. Each singlechain molecule is then cloned into a bacterial expression vector.Following amplification, one of the single-chain molecules is excisedand sub-cloned into the other vector, containing the second single-chainmolecule of interest. The resulting bscAb fragment is subcloned into aneukaryotic expression vector. Functional protein expression can beobtained by transfecting the vector into chinese hamster ovary cells.Recombinant methods can be used to produce a variety of fusion proteins.

6. Coupling of Antibodies to Lipid Emulsions

Long-circulating sub-micron lipid emulsions, stabilized withpoly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE), can beused as drug carriers for the antibodies of the present invention. Theemulsions are composed of two major parts: an oil core, e.g.,triglyceride, stabilized by emulsifiers, e.g., phospholipids. The pooremulsifying properties of phospholipids can be enhanced by adding abiocompatible co-emulsifier such as polysorbate 80. In a preferredembodiment, the antibody is conjugated to the surface of the lipidemulsion globules with a poly(ethylene glycol)-based, heterobifunctionalcoupling agent, poly(ethyleneglycol)-vinylsulfone-N-hydroxy-succinimidyl ester (NHS-PEG-VS).

The submicron lipid emulsion is prepared and characterized as described.Lundberg, J. Pharm. Sci., 83:72 (1993); Lundberg et al., Int. J. Pharm.,134:119 (1996). The basic composition of the lipid emulsion istriolein:DPPC:polysorbate 80, 2:1:0.4 (w/w). When indicated, PEG-DPPE isadded into the lipid mixture at an amount of 2-8 mol % calculated onDPPC.

The coupling procedure starts with the reaction of the NHS ester groupof NHS-PEG-VS with the amino group of distearoylphosphatidyl-ethanolamine (DSPE). Twenty-five μmol of NHS-PEG-VS arereacted with 23 μmol of DSPE and 50 μmol triethylamine in 1 ml ofchloroform for 6 hours at 40° C. to produce a poly(ethylene glycol)derivative of phosphatidyl-ethanolamide with a vinylsulfone group at thedistal terminus of the poly(ethylene glycol) chain (DSPE-PEG-VS). Forantibody conjugation, DSPE-PEG-VS is included in the lipid emulsion at 2mol % of DPPC. The components are dispersed into vials from stocksolutions at −20° C., the solvent is evaporated to dryness under reducedpressure. Phosphate-buffered saline (PBS) is added, the mixture isheated to 50° C., vortexed for 30 seconds and sonicated with a MSE probesonicator for 1 minute. Emulsions can be stored at 4° C., and preferablyare used for conjugation within 24 hours.

Coupling of antibodies to emulsion globules is performed via a reactionbetween the vinylsulfone group at the distal PEG terminus on the surfaceof the globules and free thiol groups on the antibody. Vinylsulfone isan attractive derivative for selective coupling to thiol groups. Atapproximately neutral pH, VS will couple with a half life of 15-20minutes to proteins containing thiol groups. The reactivity of VS isslightly less than that of maleimide, but the VS group is more stable inwater and a stable linkage is produced from reaction with thiol groups.

Before conjugation, the antibody is reduced by 50 mM 2-mercaptoethanolfor 10 minutes at 4° C. in 0.2 M Tris buffer (pH 8.7). The reducedantibody is separated from excess 2-mercaptoethanol with a Sephadex G-25spin column, equilibrated in 50 mM sodium acetate buffered 0.9% saline(pH 5.3). The product is assayed for protein concentration by measuringits absorbance at 280 mm (and assuming that a 1 mg/ml antibody solutionof 1.4) or by quantitation of ¹²⁵I-labeled antibody. Thiol groups aredetermined with Aldrithiol™ following the change in absorbance at 343 mmand with cystein as standard.

The coupling reaction is performed in HEPES-buffered saline (pH 7.4)overnight at ambient temperature under argon. Excess vinylsulfone groupsare quenched with 2 mM 2-mercaptoethanol for 30 minutes, excess2-mercaptoethanol and antibody are removed by gel chromatography on aSepharose CL-48 column. The immunoconjugates are collected near the voidvolume of the column, sterilized by passage through a 0.45 μm sterilefilter, and stored at 4° C.

Coupling efficiency is calculated using ¹²⁵I-labeled antibody. Recoveryof emulsions is estimated from measurements of [¹⁴C]DPPC in parallelexperiments. The conjugation of reduced LL2 to the VS group ofsurface-grafted DSPE-PEG-VS is very reproducible with a typicalefficiency of near 85%.

7. Therapeutic Use of Antibodies in Simple and Multimodal Regimens

The present invention contemplates the use of naked and/or conjugatedantibodies as the primary therapeutic composition for treatment ofautoimmune diseases. Such a composition can contain polyclonalantibodies or monoclonal antibodies. Preferred antibodies are anti-CD22antibodies, such as LL2 antibodies, including murine LL2 monoclonalantibody, chimeric LL2 antibody, and humanized LL2 antibody. Antibodiesto a single B-cell antigen or to more than one B-cell antigen may beused. In a preferred embodiment, bispecific antibodies and fusionproteins which comprise specificities for more than one B-ell antigen orepitope are employed.

For example, a therapeutic composition of the present invention cancontain a mixture of monoclonal naked anti-CD22 antibodies directed todifferent, non-blocking CD22 epitopes. Monoclonal antibodycross-inhibition studies have identified five epitopes on CD22,designated as epitopes A-E. See, for example, Schwartz-Albiez et al.,“The Carbohydrate Moiety of the CD22 Antigen Can Be Modulated byInhibitors of the Glycosylation Pathway,” in LEUKOCYTE TYPING IV. WHITECELL DIFFERENTIATION ANTIGENS, Knapp et al. (eds.), p. 65 (OxfordUniversity Press 1989). As an illustration, the LL2 antibody binds withepitope B. Stein et al., Cancer Immunol. Immunother. 37:293 (1993).Accordingly, the present invention contemplates therapeutic compositionscomprising a mixture of monoclonal anti-CD22 antibodies that bind atleast two CD22 epitopes. For example, such a mixture can containmonoclonal antibodies that bind with at least two CD22 epitopes selectedfrom the group consisting of epitope A, epitope B, epitope C, epitope Dand epitope E.

Methods for determining the binding specificity of an anti-CD22 antibodyare well-known to those of skill in the art. General methods areprovided, for example, by Mole, “Epitope Mapping,” in METHODS INMOLECULAR BIOLOGY, VOLUME 10: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.),pages 105-116 (The Humana Press, Inc. 1992). More specifically,competitive blocking assays to determine CD22 epitope specificity aredescribed by Stein et al., Cancer Immunol. Immunother. 37:293 (1993),and by Tedder et al., U.S. Pat. No. 5,484,892 (1996).

The Tedder patent also describes the production of CD22 mutants, whichlack one or more immunoglobulin-like domains. These mutant proteins wereused to determine that immunoglobulin-like domains 1, 2, 3, and 4correspond with epitopes A, D, B, and C, respectively. Thus, binding atest antibody with a panel of CD22 proteins lacking particularimmunoglobulin-like domain can also identify CD22 epitope specificity.

The therapeutic compositions described herein are useful for treatmentof autoimmune diseases, particularly for the treatment of Class IIIautoimmune diseases including immune-mediated thrombocytopenias, such asacute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myastheniagravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,polyglandular syndromes, bullous pemphigoid, diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritismultiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, Sjogren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.In this context, the therapeutic compositions are used to deplete theblood of normal B-cells for an extended period.

Although naked, preferably anti-CD22, antibodies are the preferred,primary therapeutic compositions for treatment of autoimmune diseases,the efficacy of such naked antibody therapy can be enhanced bysupplementing the naked antibodies with other therapies describedherein. In such multimodal regimens, the supplemental therapeuticcompositions can be administered before, concurrently or afteradministration of the naked, preferably anti-CD22, antibodies.Multimodal therapy of Class III autoimmune diseases may compriseco-administration of therapeutics that are targeted against T-cells,plasma cells or macrophages, such as antibodies directed against T-cellepitopes, more particularly against the CD4 and CD5 epitopes. Gammaglobulins also may be co-administered. In some cases, it may bedesirable to co-administer immunosuppressive drugs such ascorticosteroids and possibly also cytotoxic drugs. In this case, lowerdoses of the corticosteroids and cytotoxic drugs can be used as comparedto the doses used in conventional therapies, thereby reducing thenegative side effects of these therapeutics. The supplementaltherapeutic compositions can be administered before, concurrently orafter administration of the naked B-cell, preferably anti-CD22,antibodies.

In an alternative embodiment, the antibodies to the CD22, CD20, CD 19,and CD74 or HLA-DR antigen are conjugated to a drug, toxin, enzyme,hormone, cytokine, immunomodulator, boron compound or therapeuticradioisotope, or a fusion protein of an antibody and a toxin may beused. These conjugates and fusion proteins may be used alone, or incombination with naked B-cell antibodies. In a further preferredembodiment, an antibody is used that comprises an arm that is specificfor a low-molecular weight hapten to which a therapeutic agent isconjugated or fused. In this case, the antibody pretargets the B-cells,and the low-molecular weight hapten with the attached therapeutic agentis administered after the antibody has bound to the B-cell targets.Examples of recognizable haptens include, but are not limited to,chelators, such as DTPA, fluorescein isothiocyanate, vitamin B-12 andother moieties to which specific antibodies can be raised.

Drugs which are known to act on B-cells, plasma cells and/or T-cells areparticularly useful in accordance with the present invention, whetherconjugated to a B-cell antibody, or administered as a separate componentin combination with a naked or conjugated B-cell antibody. These includemethotrexate, phenyl butyrate, bryostatin, cyclophosphamide, etoposide,bleomycin, doxorubicin, carmustine, vincristine, procarbazine,dexamethasone, leucovorin, prednisone, maytansinoids such as DM1,calicheamicin, rapamycin, leflunomide, FK506, immuran, fludarabine,azathiopine, mycophenolate, and cyclosporin. Drugs such as immuran,methotrexate, and fludarabine which act on both B-cells and T-cells areparticularly preferred. Illustrative of toxins which are suitablyemployed in accordance with the present invention are ricin, abrin,ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonasendotoxin and RNAses, such as onconase. See, for example, Pastan et al.,Cell 47:641 (1986), and Goldenberg, C A—A Cancer Journal for Clinicians44:43 (1994). Other suitable drugs and toxins are known to those ofskill in the art.

Cytokine agonists and antagonists may also be used in multimodaltherapies according to the present invention. Tumor necrosis factoralpha (TNFα) and interleukin-1 (IL-1) are important in mediatinginflammation in rheumatoid arthritis. Accordingly, anti-TNFα reagents,such as Infiximab and Etanercept (Embrel), are useful in multimodaltherapy according to the invention, as well as anti-IL-1 reagents.

Other useful secondary therapeutics useful in multimodal therapies areIL-2 and GM-CSF, which may be conjugated with an anti-B-cell antibody,or combined with a naked anti-B-cell antibody as a separate component.

In general, the dosage of administered antibodies will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of antibody component,immunoconjugate or fusion protein which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate.

Administration of antibodies to a patient can be intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous,intrapleural, intrathecal, by perfusion through a regional catheter, orby direct intralesional injection. When administering therapeuticproteins by injection, the administration may be by continuous infusionor by single or multiple boluses. Intravenous injection provides auseful mode of administration due to the thoroughness of the circulationin rapidly distributing antibodies.

In preferred embodiments, naked anti-B-ell antibodies, particularlyanti-CD22 antibodies, are administered at low protein doses, such as 20milligrams to 2 grams protein per dose, given once, or repeatedly,parenterally. Alternatively, naked antibodies are administered in dosesof 20 to 1000 milligrams protein per dose, or 20 to 500 milligramsprotein per dose, or 20 to 100 milligrams protein per dose.

The antibodies, alone or conjugated to liposomes, can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic proteins are combined in a mixturewith a pharmaceutically acceptable carrier. A composition is said to bea “pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (1995).

For purposes of therapy, antibodies are administered to a patient in atherapeutically effective amount in a pharmaceutically acceptablecarrier. In this regard, a “therapeutically effective amount” is onethat is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient. In the present context, an agent isphysiologically significant if its presence results in the inactivationor killing of targeted B-cells.

Additional pharmaceutical methods may be employed to control theduration of action of an antibody in a therapeutic application. Controlrelease preparations can be prepared through the use of polymers tocomplex or adsorb the antibody. For example, biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10:1446 (1992). The rate of release ofan antibody from such a matrix depends upon the molecular weight of theprotein, the amount of antibody within the matrix, and the size ofdispersed particles. Saltzman et al, Biophys. J. 55:163 (1989); Sherwoodet al., supra. Other solid dosage forms are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th ed. (1995).

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLE 1 Treatment of a Patient with Humanized LL2

A patient undergoes therapy with humanized LL2 monoclonal antibody. Thepatient was infused intravenously with 634 mg of humanized LL2 antibody,and the treatment was repeated 6, 13, and 20 days following this initialtreatment. Immediately following the last dose, the serum value of hLL2was 389.7 μg/ml, and one month following the last dose the serum valueof hLL2 was 186.5 μg/ml. Normal B-cells in the blood prior to therapywith hLL2 were significantly depleted from the blood 2 monthspost-therapy, and there was minimal reappearance of normal B cells fivemonths post-therapy. The results are shown in the following table. TABLE1 B-cells and T-cells in blood % blood % blood % blood B-cells T-cellsHLA-Dr CD19 CD20 Kappa lambda CD3 (Ia) Day T4/T8 Flow cytometry 0 1.5 55 6 2 38 6 28 hLL2 therapy 34 hLL2 therapy 41 hLL2 therapy 48 hLL2therapy Flow cytometry 76 1.3 <2 <2 <1 <1 71 6 191 2.0 <2 <2 <1 <1 73 4

EXAMPLE 2 Treatment of a Patient with Chronic IdiopathicThrombocytopenia Purpura

A 50-year-old female with chronic idiopathic thrombocytopenia purpurahas been treated with prednisone, gamma globulins, and high dosedexamethason, but the disease progresses. She undergoes splenectomy,which fails to stabilize the disease. Her platelet count falls to lessthan 20,000/microliter, and hemorraghic events increase in frequency.The patient is then treated with hLL2, 480 mg intravenously each week,for a period of six weeks. Four weeks after the last dose of hLL2,platelet number is increased by 100%, and the hemorraghic events becomeinfrequent. Three months after the last antibody infusion the disease isin remission.

EXAMPLE 3 Treatment of a Patient with Progressive Rheumatoid Arthritis

A 60-year-old male, with severe progressive rheumatoid arthritis of thefinger joints, wrists, and elbows, has failed therapy with methotrexate,and obtains only minor relief when placed on Enbrel therapy. The patientis then treated with hLL2, 600 mg intravenously each week, for a periodof eight weeks. After 3 weeks a 30% improvement in measures of diseaseactivity is observed, which is maintained for 6 months. The patient isagain treated with hLL2, at the same dose and frequency. The patientcontinues to improve, and 6 months after the second hLL2 therapy, a 70%improvement is observed. No human anti-hLL2 antibodies are observed atany time during, or after the hLL2 therapy. Although normal B-cells aresignificantly reduced from the blood, no infectious complications, orother drug-related toxicity are observed.

EXAMPLE 4 Treatment of a Patient with Myasthenia Gravis

A 55-year-old male has failed all conventional therapy for myastheniagravis, and is admitted to a neurological intensive therapy unit. Thepatient was stabilized by plasma exchange, and given intravenousimmunoglobulin to reduce the titer of anti-acetylcholine receptorantibody. The patient remained bedridden, and was then treated withhLL2, 800 mg intravenously each week, for a period of six weeks. Oneweek after the last dose of hLL2, a 70% drop in B-lymphocytes isobserved, and a significant drop in the titer of the anti-acetylcholinewas observed. Two months after the last hLL2 dose the patient wasmobile, and was released from the hospital.

EXAMPLE 5 Combination Therapy of Progressive Rheumatoid Arthritis

Another patient with severe progressive rheumatoid arthritis of thefinger joints, wrists, and elbows, has failed therapy with methotrexate,and obtains only minor relief when placed on Enbrel therapy. The patientis then treated with 300 mg each of hLL2 and Rituximab, intravenouslyeach week, for a period of five weeks. Significant improvement inmeasures of disease activity is observed, which is maintained for 6months. The patient is again treated with the same regimen and continuesto improve. Six months after the second course of therapy, additionalimprovement is observed. No human anti-hLL2 or anti-Rituximab antibodiesare observed at any time during, or after the therapy. Although normalB-cells are significantly reduced from the blood, no infectiouscomplications, or other drug-related toxicity are observed.

EXAMPLE 6 Combination Therapy of Chronic Idiopathic ThrombocytopeniaPurpura

A patient with chronic idiopathic thrombocytopenia purpura has beentreated with prednisone, gamma globulins, and high dose dexamethason,but the disease progresses. He undergoes spleenectomy, which fails tostabilize the disease. The platelet count falls to less than20,000/microliter, and hemorraghic events increase in frequency. Thispatient is treated with 10 mCi of 90-yttrium-hLL2 and 200 mg of hLL2,followed by 300 mg doses each of hLL2 and Rituximab, intravenously eachweek, for a period of six weeks. Four weeks after the last dose of hLL2and Rituximab, platelet number is increased by 150%, and the hemorraghicevents become infrequent. Three months after the last antibody infusionthe disease is in remission.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those in the art to which theinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference in its entirety.

1. A method of treating an autoimmune disease in a subject comprisingadministering an anti-CD20 antibody and an anti-TNFα antagonist oranti-IL-1 antagonist.
 2. A method according to claim 1, wherein saidanti-TNFα or anti-IL-1 antagonist is administered before said anti-CD20antibody.
 3. A method according to claim 1, wherein said anti-TNFα oranti-IL-1 antagonist is administered after said anti-CD20 antibody.
 4. Amethod according to claim 1, wherein said anti-TNFα or anti-IL-1antagonist is administered concurrently with said anti-CD20 antibody. 5.A method according to claim 1, comprising administering an anti-CD20antibody and an anti-TNFα antagonist.
 6. A method according to claim 5,wherein said anti-TNFα antagonist is administered before said anti-CD20antibody.
 7. A method according to claim 5, wherein said anti-TNFαantagonist is administered after said anti-CD20 antibody.
 8. A methodaccording to claim 5, wherein said anti-TNFα antagonist is administeredconcurrently with said anti-CD20 antibody.
 9. A method according toclaim 1, comprising administering an anti-CD20 antibody and an anti-IL-1antagonist.
 10. A method according to claim 5, wherein said anti-IL-1antagonist is administered before said anti-CD20 antibody.
 11. A methodaccording to claim 5, wherein said anti-IL-1 antagonist is administeredafter said anti-CD20 antibody.
 12. A method according to claim 5,wherein said anti-IL-1 antagonist is administered concurrently with saidanti-CD20 antibody.
 13. A method according to claim 1, wherein saidanti-TNFα antagonist is an anti-TNFα antibody and said anti-IL-1antagonist is an anti-IL-1 antagonist is an anti-IL-1 antibody.
 14. Amethod according to claim 1, comprising administering an anti-CD20antibody and an anti-TNFα antibody.
 15. A method according to claim 1,comprising administering an anti-CD20 antibody and an anti-IL-1antibody.
 16. A method according to claim 1, wherein said autoimmunedisease is rheumatoid arthritis.
 17. A method according to claim 14,wherein said autoimmune disease is rheumatoid arthritis.
 18. A methodaccording to claim 15, wherein said autoimmune disease is rheumatoidarthritis.